For several decades equilibrium dialysis techniques were the only available method for the measurement of free hormones in serum, and until recently were the only methods considered reliable. Equilibrium dialysis methods in this context suffer from several drawbacks including poor precision, tediousness and so on; but above all their results are highly dependent on the purity of the tracers used.
Ellis and Ekins, R. (Acta Endocr. (KbH.) Suppl. 177:106, 1973), disclosed a direct method for free hormone determinations in their paper xe2x80x9cDirect Measurement By Radioimmunoassay of the Free Thyroid Hormone Concentration in Serium.xe2x80x9d This represented a major improvement over equilibrium dialysis methods because it allowed for the direct measurement by radioimmunoassay (RIA) of free ligand levels in serum dialysates, thus circumventing the problem of tracer purity. This method is now considered by many as the reference methodology for CO free hormone measurements. It is, however, still time consuming and operator-dependent, and it is unavailable to most small laboratories.
Indirect methods for the estimation of free hormone concentrations which were introduced shortly thereafter include the testosterone/steroid hormone binding globulin (SHBG) ratio, the thyroxine (T4)/thyroid binding globulin (TBG) ratio, the free T4 index (based on the product of triiodothyronine (T3) uptake and T4), and the free androgen index.
Ekins, R. (Free Thyroid Hormones; Proceedings of the International Symposium held in Venice, December 1978, Amsterdam: Excerpta Medica, 1979 72-92), introduced the concept of xe2x80x9cdirect dynamic methodsxe2x80x9d in which an anti-free ligand antibody is used in direct contact with the biological fluid during dialysis. This constitutes the basis for so-called xe2x80x9cimmunoextractionxe2x80x9d methods.
One such method is taught in U.S. Pat. No. 4,046,870 in which a two-tube immunoassay method measures the rate of transfer of T4 from binding proteins to T4-specific antibody. This method suffered from several analytical and clinical shortcomings which made it virtually just another free T4 index assay.
A second method, introduced by Clinical Assays (Cambridge, Mass. 02139), was a true immunoextraction method. It used a single-tube, two-stage, sequential (back-titration) technique. In this method, a serum sample is incubated with immobilized antibody; then, following a wash step, unoccupied sites on the immobilized antibody are xe2x80x9cback-titratedxe2x80x9d using labeled ligand. In this approach, the serum is never in contact with the labeled ligand. Although theoretically sound, it suffers from poor sensitivity and precision, and both reactions require exact timing.
Single-step immunoextraction methods for the determination of free ligand concentrations in biological specimens were the obvious next step in the development of free ligand assay systems. These methods rely on chemical rather than physical separation of labeled ligand from nedogenous binders. In order to achieve this objective, several approaches can be adopted, as detailed below.
The prior art discloses that by chemically altering the structure of a given ligand, its binding to endogenous binders is reduced or diminished. This has been amply demonstrated for steroid hormones. (See the discussion of free testosterone below.) In the case of thyroid hormones, Ross, J. E. and Tapley, D. F. (Effect of various analogues on the binding of labeled thyroxine to thyroxine-binding globulin and prealbumin, Endocrinology 79:493, 1966), have shown that the binding of TBG (thyroid binding globulin) to T4 is inhibited if a fairly bulky substitution is made at the 3xe2x80x2 position of the T4 molecule. In addition, Schall, R. F., et al (An enzyme-labeled immunoassay for the measurement of unsaturated thyroid hormone binding capacity in serum and plasma, Clin. Chem. 25:1078 (abstract) 1979), and Kleinhammer, G., et al (Enzyme immunoassay for determination of thyroxine binding index, Clin. Chem. 24:1033, 1978), independently demonstrated that TBG fails to bind to conjugates formed by labeling T4 with horseradish peroxidase. This fact constitutes the basis for the single-step immunoextraction method described in U.S. Pat. No. 4,410,633 to Corning Glass Works, for the measurement of free thyroxine and free 3,5,3xe2x80x2-triiodothyronine wherein horseradish peroxidase is chemically attached to T4 and T3 and later radiolabeled.
In addition, the prior art also discloses that T3 and T4 require the following molecular structure for maximal binding to endogenous binding proteins, viz. TBG, thyroid binding pre-albumin (TBPA), albumin, Snyder, S. M, et al (Binding of thyroid hormones and their analogues to thyroxine-globulin in human serum, J. Biol. Chem. 251:6489, 1976); Sterling, K., et al (Equilibrium dialysis studies of the binding of thyroxine by human serum albumin, J. Clin. Invest. 41:1021, 1962):
1. The L-alanine side chain configuration:
2. The presence of 4xe2x80x2-hydroxyl group (primarily for TBPA and albumin binding); and
3. The presence of two (halogen) substituents in the inner and outer rings (positions 3,5,3xe2x80x2 and 5xe2x80x2).
Several hundred T3 and T4 analogs have been synthesized and studied for their ability to bind to thyroid hormone binding proteins.
U.S. Pat. No. 4,366,143 and its European counterpart, Patent No. 00 26 103, broadly describe the use of such analogs as tracers in a single immunoextraction using simultaneous rather than sequential titration of antibody for the measurement of free hormones. (For convenience, these patents will be collectively referred to hereinafter as the xe2x80x9cAmershamxe2x80x9d patent.)
An intact alanine side chain is required for optimal binding of T4 and T3 to TBG: the amino group on the analine side chain is the essential constituent. Analogs described in the Amersham patent are T3 and T4 molecules modified at the alanine side chain. Although theoretically these analogs do not bind TBG to any significant extent, they undoubtedly bind albumin and TBPA significantly since the 4xe2x80x2-hydroxyl group on the T3 and the T4 molecules is left intact. It is well established that the binding of albumin and TBPA to the thyronines is quantitative, especially under physiological conditions, Sterling, K. (Molecular structure of thyroxine in relation to its binding by human serum albumin, J.Clin Invest. 43:1721, 1964), and Pages, et al (Binding of thyroxine and thyroxine analogs to human serum prealbumin, Biochem 12:2773, 1973).
The failure of the Amersham patent to recognize the importance of albumin and TBPA binding to the thyronines renders the patent""s teachings inadequate for the true measurement of free T3 and free T4 in biological fluids. In fact the commercially available reagents eased on the patent yield misleading and inaccurate free hormone results. This is particularly true in several pathological conditions characterized by significant alterations in the circulating albumin level.
Recent literature has shown that the albumin concentration correlates directly with free T4 concentrations generated by the Amersham assay system. In addition, it is well documented that Amersham""s method consistently yields falsely decreased free T4 results in third-trimester pregnancies and in patients suffering from severe non-thyroidal illness, while yielding falsely elevated free T4 levels in cases of familial dysalbuminemic hyperthyroxinemia, a condition in which T4 is abnormally bound to circulating albumin.
During pregnancy, albumin circulates at lower than normal levels, especially during the third trimester. Since Amersham""s labeled analog T4 tracer binds albumin and TBPA to a significant extent (greater than 99%), one would expect the Amersham assay system to yield lower than normal free T4 results during the third trimester: more analog tracer is available to bind T4 antibody, resulting in higher binding and lower apparent dose.
Non-esterified free fatty acids are capable of displacing labeled analog from albumin; moreover, they circulate at higher than normal concentrations during pregnancy. This could explain the lower than expected free T4 values encountered during pregnancy when assayed by the Amersham method; apparent free T4 levels would be significantly lower than expected if albumin binding to the labeled analog is substantial.
This Situation is also well documented in cases of heparin therapy, where a significant elevation of non-esterified free fatty acids is present. Free T4 and free T3 levels when measured by Amersham""s method on heparin-treated patients show lower than normal levels.
The same problem occurs for non-thyroidal illness, where free T3 and T4 values generated by the Amersham method have been shown to be significantly lower than for a euthyroid population, when compared to a direct equilibrium dialysis method.
The Amersham patent procedure has been found wanting by workers in the art as manifested by the observance of false and erroneous measurements of free ligand levels. Applicant has discovered that the problem stems from binding of the ligand analog tracer to certain endogenous proteins, e.g., albumin in biological fluids. I have discovered that this problem can be overcome by the use of specific chemical inhibitor reagents. This discovery represents a major advance in the art and it is believed to be deserving of a patent.
Briefly, this invention comprises a method for measuring free ligands in biological fluids in the presence of bound ligand and endogenous binding proteins, without disturbing the equilibrium between the free ligand and the protein-bound ligand, comprised of the following steps: (a) incubating a sample of biological fluid with (i) a ligand analog tracer which, due to its chemical structure, does not bind to some of the endogenous binding proteins, (ii) a specific ligand binder and (iii) at least one specific chemical inhibitor reagent that inhibits the binding of the ligand analog tracer to other endogenous binding proteins; (b) separating the ligand analog tracer bound to the specific binder from unbound tracer; and (c) determining the concentration of free ligand in said biological fluid.
It is an object of this invention to provide a new and improved method for measuring free ligands in biological fluids.
More particularly, the present invention has as its object the truer measurement of free ligands in biological fluids.
These and other objects and advantages of my invention will be apparent from the detailed description which follows.
The present invention addresses the deficiencies encountered in the Amersham patent and effectively corrects for the inconsistencies in free thyroid results generated by Amersham""s analog method.
The present invention uses labeled analogs for T3 and T4 that are modified at the analine side chain. Specifically, the xcex1-amino group is modified to prevent their binding to TBG. Meanwhile, steps have been taken to prevent such labeled analogs from binding to albumin and TBPA. This is accomplished by carefully selecting an exogenous chemical reagent or reagents that alone or in combination are able to bind to unoccupied binding sites on the albumin and TBPA molecules, thus saturating these binding proteins and effectively eliminating their capacity to bind to thyronine analogs and to other endogenous substances such as non-esterified free fatty acids. These exogenous chemicals should not bind to TBG and their concentration should be such as not to displace any bound hormone from albumin or TBPA.
The association constant for albumin and T4 is approximately 500,000. (This estimate is based on the assumption that the number of binding sites on the albumin molecule available for thyroxine is equal to 1, and that the apparent association constant in liters per molexe2x80x94i.e. the equilibrium constant in the direction of complex formationxe2x80x94is 5xc3x97105.) Likewise, the association constant for albumin and T3 is approximately 24,600. It is well established that albumin has a higher affinity for free T3 and T4 and their analogs than for anionic dyes, but a much higher affinity for free fatty acids than T3 and T4 and their analogs.
Albumin has a relatively low association constant for single aromatic compounds; the highest association constants are for 2,4-dinitrophenol (11,000) and salicylate (2,800).
In order to maintain strict equilibrium conditions in vitro during the immunoextraction reaction one has to maintain strict physiological conditions; this entails the use of pH=7.4. At that pH, thyronine molecules have three charged groups: the anionic carboxylate ion, the cationic xcex1-amino group and the anionic phenolate ion. (The latter is 82% ionized.) The presence of albumin or TBPA under these physiological conditions yields a highly charged albumin with a relatively large number of catiohic amino groups. These cationic amino groups on the albumin molecule bind the anionic phenolate ion on the thyronine molecules. Such an interaction is the main cause of albumin binding to the labeled analog in both the Amersham patent method and the Corning patent method.
The present invention makes use of the fact that 2,4-dinitrophenol (DNP) and sodium salicylate with their relatively high association constants to albumin and TBPA will also be ionized and charged under these physiological conditions of pH, yielding charged anionic phenolate ion capable of interaction with the charges on the albumin and TBPA molecules. When either 2,4-dinitrophenol or sodium salicylate or both are present in excess, the binding of labeled T3 and T4 analogs to albumin and TBPA is virtually eliminated. This method of blocking albumin and TBPA by appropriate concentrations of 2,4-dinitrophenol and/or sodium salicylate is an effective means for eliminating the erroneous assay results caused by albumin in free thyroid hormone immunoex-traction analog methods.
The present invention is applicable to a variety of other chemical inhibitor reagents, that is, reagents capable of blocking unwanted reaction of the ligand analog tracer to circulating endogenous binding proteins. The substituted monoaryl organic compounds are exemplary. The substituents on such compounds include nitro, carboxyl, carboxyl salts and the like. The monoaryl compounds have a phenolic hydroxyl group which are particularly useful. Another suitable category are the dyes such as sulfobromophthalein, orange red, bromocresyl blue and the like. The higher (over about 5 carbon atoms) fatty acids such as oleic acid are also useful. Still other compounds will be apparent to those skilled in the art. For example, many amino acids have a high affinity to albumin to and hence are useful in the practice of this invention, e.g., tryptophan. Another suitable category are T3, T4 or testosterone analogs which displace labeled analog from endogenous proteins while not binding to the antibody or other specific ligand binder.
This invention can be used to detect the concentration of any of the free ligands normally found in human body fluid. For example, the free ligand can be thyroxine, tri-iodothyroxine, testosterone, cortisol, progesterone, oestradiol, hormones and steroids generally, also drugs and products of drug metabolism, vitamins such as B12, toxins, and the like.
In general, specific ligand binder is one which couples or binds to the free ligand and it may be a specific antibody for the free ligand or other binding agent. In general, the specific ligand binders appropriate to the various free ligands are known and need not be further described.
The ligand analog tracer is labeled in some way so as to be detectable or observable. Radiolabels are well-known and applicable, as are the other labeling means previously employed in this art, including enzymes, fluorophors, chromophores and chemiluminescent groups integral with the ligand analog tracer molecule.